Composition comprising a prebiotic for decreasing inflammatory process and abnormal actibation of non-specific immune parameters

ABSTRACT

The present invention relates to a composition comprising prebiotic (prebiotic adjuvant) for decreasing inflammatory process by improving the homeostasis of non-specific immune parameters and of lymphocyte subpopulations. It also relates to the use of a prebiotic formulation in the manufacture of a medicament or a food or petfood composition for decreasing inflammatory process and/or abnormal activation of non-specific immune parameters, such as phagocytes.

FIELD OF THE INVENTION

[0001] The present invention relates to a composition comprisingprebiotic (prebiotic adjuvant) for decreasing inflammatory process byimproving the homeostasis of non-specific immune defense parameters andof lymphocyte subpopulations. It also relates to the use of a prebioticformulation in the manufature of a medicament or food or petfoodcomposition for decreasing inflammatory process and/or abnormalactivation of non-specific immune parameters, such as phagocytes.

BACKGROUND OF THE INVENTION

[0002] It is well known that prebiotics comprise carbohydrates and morespecifically, oligosaccharides. Furthermore it is known that they havewidely been used as functional food ingredients. They resist hydrolysisby enzymes of the human digestive tract, can reach the colon undegradedand provide a carbohydrate substance particulary suited to the growth ofbifidobacteria Oligosaccharides may be produced from glucose, galactose,xylose, maltose, sucrose, lactose, starch, xylan, hemicellulose, inulin,gum or a mixture thereof. Purified commercially available products suchas fructooligosaccharides contain greater than about 95% solids in theform of oligosaccharides.

[0003] Fructooligosaccharides have been studied in humans mainly forfunctional claims related to the bioavailability of minerals, lipidmetabolism and and regulation of bowel habits (Roberfroid, M. B.Delzenne, N. M. Annu Rev Nutr 1998;18:117-143). Little attention hasbeen given to their effect on immunological functions, while indicationsfor modifications of carcinogenesis and stimulation of gut-associatedlymphoid tissue comes from animal studies (Pierre, F., et al. Cancer Res1997;57:225-228) .

[0004] Indeed, fructooligosaccharides, long (inulin) and short chain(oligofructose) are among the carbohydrates, which escape digestion inthe upper gastro-intestinal tract. They are then fermented in the colonand selectively stimulate the growth of bifidobacteria.

[0005] Human intestinal flora with its important metabolic activity ispossibly associated with many health related functions such asmaintenance of gut homeostasis, metabolism of xenobiotics andstimulation of gut immunity. It is influenced by disease, diet, stressand possibly ageing. The large intestine contains up to 10¹² bacteria/gfaeces with about 10³ different species from approximately 40-50 generaof bacteria. Most of them are obligate anaerobes with a largepopulation, however, of facultative anaerobes. The main anaerobe speciesare Bacteroïdes, bifidobacteria, eubacteria, which make up to 99% of thetotal faecal flora, followed by clostridia, lactobacilli and grampositive cocci, enterococci, coliforms, methanogens and at much lowerlevels sulfate-reducing bacteria (Hill, M. J. Normal gut bacterialflora. 1995;3-17).

[0006] Adult microflora characteristics are present from about 2 yearsof age. Adult gut microflora seems to be rather stable; although somechanges have been reported with ageing, mainly low levels ofbifidobacteria and Bacteroïdes (Hopkins, M. J., et al. Gut2001;48:198-205). Gut flora can be divided into species that havebeneficial effects, such as bifidobacteria, or harmful effects, such asPseudonioinas aeruginosa, Protezus species, staphylococci, someclostridia and Veilonellae, and species that are intermediate in effectsuch as enterococci, Escherichia coli, Enterococci and Bacteroïdes.Bifidobacteria and lactobacilli have been reported to have beneficialeffects on specific immune functions (Schiffrin, E. J., et al. J DairySci 1995;78:491-497).

[0007] With age it is generally reported that bifidobacteria arediminished, while Clostridim perfringens, Entercocci andEnterobacteriaceae are increased (Mitsuoka, T. Hayakawa, K. ZentralblBakteriol [Orig A] 1973;223:333-342). Bacterial overgrowth occurs morefrequently in the elderly due to the high prevalence of atrophicgastritis and hypochlorhydria. Bacterial overgrowth seems to be free ofclinical symptoms in healthy elderly, it may have some importance infrail elderly ≧75 year of age, and Clostridium difficile associateddiarrhea is more frequent in the elderly in acute care or long-termcare, in association with antibiotic treatment and possibly decreasedimmune response. Ageing is related with a loss in immune function andthe existence of an interrelationship between nutrition and immunefunction has been recognized (Meydani, S. N. Status of NutritionalImmunology Studies: J Nutr Immunol 1994;2:93-97).

[0008] Changes in immune response (remodeling of cytokine production anddysregulation of the immune functions) is associated with increasedincidence of infections and mortality linked to infection. Nutritionalinterventions, mainly vitamin and mineral supplementation, can improvethe immune response in frail elderly [Lesourd, B. M. Am J Clin Nutr1997;66:478S-484S41].

[0009] The present invention aims to provide another composition able tolimit the dysregulation of the immune function, and more particularlythe abnormal activation of non-specific immune response such as thephagocytes and the monocyte macrophage cell system as well as preservelymphocyte subpopulations in normal level of activation.

SUMMARY OF THE INVENTION

[0010] Consequently, in a first aspect the present invention provides acomposition comprising at least one prebiotic for decreasinginflammatory process and/or abnormal activation of non-specific immuneparameters.

[0011] It may be particularly intended for decreasing abnormalactivation of phagocytes, for example.

[0012] It has been surprinsingly found that a prebiotic supplementationcan induce decrease in inflammatory process, and particularly can inducechanges in non-specific immunity, such as decreased phagocytic activity,as well as a decreased expression of interleukin-6 mRNA in peripheralblood monocytes.

[0013] In a second aspect the invention provides the use of at least oneprebiotic in the manufature of a medicament or a food or pet foodcomposition for decreasing inflammatory process in a mammal.

[0014] In a third aspect the invention provides use of at least oneprebiotic in the manufature of a medicament or a food or pet foodcomposition for decreasing abnormal activation of non-specific immuneparameters in a mammal.

[0015] In a forth aspect the invention provides a method of decreasinginflammatory process in a mammal, which comprises administering aneffective amount of a prebiotic or composition comprising at least oneprebiotic.

[0016] In a fifth aspect the invention provides a method of decreasingabnormal activation of non-specific immune parameters in a mammal, whichcomprises administering an effective amount of a prebiotic orcomposition comprising at least one prebiotic.

[0017] An advantage of the present invention is that it provides adecrease in inflammatory process, particularly a decrease expression ofInterleukine-6 mRNA in peripheral blood mononuclear cells.

[0018] Another advantage of the present invention is that it provides adecrease in phagocytic activity of granulocytes and monocytes,particularly in frail patient with chronic inflammatory situation.

[0019] Yet another advantage of the present invention is that it may beused to improve the inflammatory situation in a mammal and thus reducethe risk of development of deleterious infections, by simple consumptionof a food composition according to the present invention. It will beappreciated that intravenous or subcutaneous administration of a drugrequires expertise, and compared to oral administration it is not assafe, convenient or acceptable to the patient. In the light of theseconcerns, the invention provides the clear advantage of a nutritionaland/or therapeutic product which may be administered orally.

DETAILED DESCRIPTION

[0020] According to a first aspect, the composition preferably comprisesat least one prebiotic or a prebiotic mixture.

[0021] Preferably, the prebiotic comprises an oligosachharide producedfrom glucose, galactose, xylose, maltose, sucrose, lactose, starch,xylan, hemicellulose, inulin, gum (acacia gum, for example) or a mixturethereof. More preferably the oligosaccharide comprisesfructooligosaccharide (FOS). Most preferably the prebiotic comprises amixture of fructooligosaccharide and inulin. Preferably this mixturecomprises PREBIO1® or a mixture of commercially available RAFTILOSE® andRAFTILINE®.

[0022] Preferably, the prebiotic comprises about 50% to about 95% FOS.More preferably it comprises about 60% to about 80% FOS. Most preferablyit comprises about 70% FOS.

[0023] Preferably, the prebiotic comprises about 10% to about 50%inulin. More preferably it comprises about 20% to about 40% inulin. Mostpreferably it comprises about 30% inulin.

[0024] The prebiotic may comprise a mixture of fructooligosaccharidesand inulin in the amounts by weight of 70% fructooligosaccharides and30% inulin.

[0025] Preferably, the composition comprises a probiotic in addition tothe prebiotic. The probiotic may be Bifidobacterium bifidum orStreptococcus thermophilus, for example. Preferably the Bifidobacteriumbifidum is Bifidobacterium lactis.

[0026] In one embodiment, the composition may be a complete andnutritionally balanced food or pet food. It can also be a dietarysupplement, for example. It is preferably adressed to elderly human orelderly pet, or critically ill patients with chronic inflammation.

[0027] Accordingly, a nutritionally complete pet food can be prepared.The nutritionally complete pet food may be in any suitable form; forexample in dried form, semi-moist form or wet form; it may be a chilledor shelf stable pet food product. These pet foods may be produced as isconventional. Preferably, the prebiotic is provided in the form of plantmaterial, which contains the prebiotic. Suitable plant materials includeasparagus, artichokes, onions, wheat, yacon or chicory, or residues ofthese plant materials. Alternatively, the prebiotic may be provided asan inulin extract or its hydrolysis products commonly known asfructooligosaccharides, galacto-oligosaccarides, xylo-oligosaccharidesor oligo derivatives of starch. Extracts from chicory are particularlysuitable. The maximum level of prebiotic in the pet food is preferablyabout 20% by weight; especially about 10% by weight. For example, theprebiotic may comprise about 0.1% to about 5% by weight of the pet food.For pet foods which use chicory as the prebiotic, the chicory may beincluded to comprise about 0.5% to about 10% by weight of the feedmixture; more preferably about 1% to about 5% by weight.

[0028] Apart from the prebiotic according to the invention, these petfoods may include any one or more of a carbohydrate source, a proteinsource and lipid source.

[0029] Any suitable carbohydrate source may be used. Preferably thecarbohydrate source is provided in the form of grains, flours andstarches. For example, the carbohydrate source may be rice, barley,sorghum, millet, oat, corn meal or wheat flour. Simple sugars such assucrose, glucose and corn syrups may also be used. The amount ofcarbohydrate provided by the carbohydrate source may be selected asdesired. For example, the pet food may contain up to about 60% by weightof carbohydrate.

[0030] Suitable protein sources may be selected from any suitable animalor vegetable protein source; for example muscular or skeletal meat, meatand bone meal, poultry meal, fish meal, milk proteins, corn gluten,wheat gluten, soy flour, soy protein concentrates, soy protein isolates,egg proteins, whey, casein, gluten, and the like. For elderly animals,it is preferred for the protein source to contain a high quality animalprotein. The amount of protein provided by the protein source may beselected as desired. For example, the pet food may contain about 12% toabout 70% by weight of protein on a dry basis.

[0031] The pet food may contain a fat source. Any suitable fat sourcemay be used both animal fats and vegetable fats. Preferably the fatsource is an animal fat source such as tallow. Vegetable oils such ascorn oil, sunflower oil, safflower oil, rape seed oil, soy bean oil,olive oil and other oils rich in monounsaturated and polyunsaturatedfatty acids, may also be used. In addition to essential fatty acids(linoleic and alpha-linoleic acid) the fat source may include long chainfatty acids. Suitable long chain fatty acids include, gamma linoleicacid, stearidonic acid, arachidonic acid, eicosapentanoic acid, anddocosahexanoic acid. Fish oils are a suitable source of eicosapentanoicacids and docosahexanoic acid. Borage oil, blackcurrent seed oil andevening primrose oil are suitable sources of gamma linoleic acid.Rapeseed oil, soybean oil, linseed oil and walnut oil are suitablesources of alpha-linoleic acid. Safflower oils, sunflower oils, cornoils and soybean oils are suitable sources of linoleic acid. Olive oil,rapeseed oil (canola) high oleic sunflower and safflower, peanut oil,rice bran oil are suitable sources of monounsaturated fatty acids. Theamount of fat provided by the fat source may be selected as desired. Forexample, the pet food may contain about 5% to about 40% by weight of faton a dry basis. Preferably, the pet food has a relatively reduced amountof fat.

[0032] The pet food may contain other active agents such as long chainfatty acids. Suitable long chain fatty acids include alpha-linoleicacid, gamma linoleic acid, linoleic acid, eicosapentanoic acid, anddocosahexanoic acid. Fish oils are a suitable source of eicosapentanoicacids and docosahexanoic acid. Borage oil, blackcurrent seed oil andevening primrose oil are suitable sources of gamma linoleic acid.Safflower oils, sunflower oils, corn oils and soybean oils are suitablesources of linoleic acid.

[0033] The choice of the carbohydrate, protein and lipid sources is notcritical and will be selected based upon nutritional needs of theanimal, palatability considerations, and the type of product produced.Further, various other ingredients, for example, sugar, salt, spices,seasonings, vitamins, minerals, flavoring agents, gums, and probioticmicro-organisms may also be incorporated into the pet food as desired

[0034] A probiotic microorganism may also be added. It may be selectedfrom one or more microorganisms suitable for animal consumption andwhich is able to improve the microbial balance in the intestine.Examples of suitable probiotic micro-organisms include yeast such asSaccharomyces, Debaromyces, Candida, Pichia and Torulopsis, moulds suchas Aspergillus, Rhizopus, Mucor, and Penicillim and Torulopsis andbacteria such as the genera Bifidobacterium, Bacteroides, Clostridium,Fusobacterium, Melissococcus, Propionibacterium, Streptococcus,Enterococcus, Lactococcus, Staphylococcus, Peptostrepococcus, Bacillus,Pediococcus, Micrococcus, Leuconostoc, Weissella, Aerococcus, Oenococcusand Lactobacillus. The probiotic micro-organisms may be in powdered,dried form; especially in spore form for micro-organisms which formspores. Further, if desired, the probiotic micro-organism may beencapsulated to further increase the probability of survival; forexample in a sugar matrix, fat matrix or polysaccharide matrix. If aprobiotic micro-organism is used, the pet food preferably contains about10⁴ to about 10¹⁰ cells of the probiotic micro-organism per gram of thepet food; more preferably about 10⁶ to about 10⁸ cells of the probioticmicro-organism per gram. The pet food may contain about 0.5% to about20% by weight of the mixture of the probiotic micro-organism; preferablyabout 1% to about 6% by weight; for example about 3% to about 6% byweight.

[0035] For elderly pets, the pet food preferably contains proportionallyless fat than pet foods for younger pets. Further, the starch sourcesmay include one or more of oat, rice, barley, wheat and corn.

[0036] For dried pet foods a suitable process is extension cooking,although baking and other suitable processes may be used. When extrusioncooked, the dried pet food is usually provided in the form of a kibble.If a prebiotic is used, the prebiotic may be admixed with the otheringredients of the dried pet food prior to processing. A suitableprocess is described in European patent application No 0850569;. If aprobiotic micro-organism is used, the organism is best coated onto orfilled into the dried pet food. A suitable process is described inEuropean patent application No 0862863.

[0037] For wet pet foods, the processes described in U.S. Pat. Nos.4,781,939 and 5,132,137 may be used to produce simulated meat products.Other procedures for producing chunk type products may also be used; forexample cooking in a steam oven. Alternatively, loaf type products maybe produced by emulsifying a suitable meat material to produce a meatemulsion, adding a suitable gelling agent, and heating the meat emulsionprior to filling into cans or other containers.

[0038] In another embodiment, a food composition for human consumptionis prepared. This composition may be a nutritional complete formula, adairy product, a chilled or shelf stable beverage, soup, a dietarysupplement, a meal replacement, and a nutritional bar or aconfectionery.

[0039] Apart from the prebiotic according to the invention, thenutritional formula may comprise a source of protein. Dietary proteinsare preferably used as a source of protein. The dietary proteins may beany suitable dietary protein; for example animal proteins (such as milkproteins, meat proteins and egg proteins); vegetable proteins (such assoy protein, wheat protein, rice protein, and pea protein); mixtures offree amino acids; or combinations thereof. Milk proteins such as casein,whey proteins and soy proteins are particularly preferred. Thecomposition may also contain a source of carbohydrates and a source offat.

[0040] If the nutritional formula includes a fat source, the fat sourcepreferably provides about 5% to about 55% of the energy of thenutritional formula; for example about 20% to about 50% of the energy.The lipids making up the fat source may be any suitable fat or fatmixtures. Vegetable fats are particularly suitable; for example soy oil,palm oil, coconut oil, safflower oil, sunflower oil, corn oil, canolaoil, lecithins, and the like. Animal fats such as milk fats may also beadded if desired.

[0041] A source of carbohydrate may be added to the nutritional formula.It preferably provides about 40% to about 80% of the energy of thenutritional composition. Any suitable carbohydrates may be used, forexample sucrose, lactose, glucose, fructose, corn syrup solids, andmaltodextrins, and mixtures thereof. Dietary fibre may also be added ifdesired. If used, it preferably comprises up to about 5% of the energyof the nutritional formula. The dietary fibre may be from any suitableorigin, including for example soy, pea, oat, pectin, guar gum, gumarabic, and fructooligosaccharides. Suitable vitamins and minerals maybe included in the nutritional formula in an amount to meet theappropriate guidelines.

[0042] One or more food grade emulsifiers may be incorporated into thenutritional formula if desired; for example diacetyl tartaric acidesters of mono- and di-glycerides, lecithin and mono- and di-glycerides.Similarly suitable salts and stabilisers may be included.

[0043] The nutritional formula is preferably enterally administrable;for example in the form of a powder, tablet, capsule, a liquidconcentrate, solid product or a ready-to-drink beverage. If it isdesired to produce a powdered nutritional formula, the homogenisedmixture is transferred to a suitable drying apparatus such as a spraydrier or freeze drier and converted to powder.

[0044] In another embodiment, a nutritional composition comprises a milkbased cereal together with a prebiotic formulation. Preferably the milkbased cereal is an infant cereal which acts as a carrier for theprebiotic formulation.

[0045] In another embodiment, a usual food product may be enriched withat least one prebiotic according to the present invention. For example,a fermented milk, a yoghurt, a fresh cheese, a renneted milk, article ofconfectionery, for example a sweet or sweetened beverage, aconfectionery bar, breakfast cereal flakes or bars, drinks, milkpowders, soy-based products, non-milk fermented products or nutritionalsupplements for clinical nutrition. Then, the amount of the compositionadded is preferably at least about 0.01% by weight.

[0046] The following examples are given by way of illustration only andin no way should be construed as limiting the subject matter of thepresent application. Percentages and parts are by weight unlessotherwise indicated. The example are preceeded by a brief description ofthe figures.

[0047]FIG. 1: Effect of feeding 8 g of short-chainfructooligosaccharides (FOS) on viable counts of bifidobacteria in freshfaecal samples of nursing home elderly. Time points were before (2),during (3), and after (4) period of 3 weeks of FOS intake. (A)Individual values. (B) Box plots. Boxes indicate 25^(th) and 75^(th)percentiles, solid lines inside box indicate median values, and t-shapes5^(th) and 95^(th) percentiles; solid dot indicates outlying value.

[0048]FIG. 2:: Effect of fructooligosaccharides (FOS) on bifidobacteriain adults and elderly. Reference 24 (Bouhnik Y et al. J Nutr 1999;129:113) dose response from 2.5 to 20 g FOS/day in 18-47 year oldadults; Reference 25 (Menne E et al. J Nutr 2000;130:1197) response to 8g FOS/day in 20-50 year old adults; Reference 26 (Gibson G R J Nutr1999;129:1438S) response to 15 gFOS/day in young adults; Reference 27(Kruse H P et al. Brit J Nutr 1999;82:375) response to inulin up to 34g/day in 26-53 year old adults; Reference 28 (Kleessen B et al. Am JClin Nutr 1997; 65:1397) response to 20 g FOS/day in elderly constipatedsubjects aged 68-89 years; Present study: resposne to 8 g FOS/day innursing home subjects aged 77-91 years.

[0049]FIG. 3: Differences in IL-6 mRNA expression in peripheral bloodmononuclear cells (PBMC) of elderly supplemented with FOS. IL-6 mRNAexpression in PBMC was measured by reverse transcription-polymerasechain reaction (RT-PCR). Estimation of quantitative changes were done byscanning band intensity using NIH-image program and calculated asdensity ratio of IL-6 mRNA to β-actin mRNA in percent. Time points werebefore (2), during (3), and after (4) period of 3 weeks of FOS intake.(A) Representative image of individual changes from ethidium bromidestained gels. (B) Quantitative estimation of IL-6 mRNA expression:Changes in IL6-mRNA during FOS intakes are significantly different frombefore FOS intakes (p=0.018).

EXAMPLE 1 Effects of Oligosaccharide on the Faecal Flora AndNon-Specific Immune System in Elderly People

[0050] Materials and Methods

[0051] Study Design

[0052] The study was a pretest/posttest study of 19 elderly nursing homepatients (see study scheme).

[0053] Measure of body weight and sample collection for stool, blood andurine were at time point 1 (pretest or at the beginning of the wash outperiod), 2 (before prebiotic intake), 3 (during prebiotic intake) and 4(after follow up period)

[0054] During the whole study, intake of fermented dairy products wererestricted and fructooligosaccharides containing food (onion, leek,chicory roots) were limited.

[0055] Subjects

[0056] Nineteen elderly subjects in a nursing home were recruited forthe study. Subjects fulfilling one or more of the following criteriawere excluded from participation in the study:

[0057] Antibiotic treatment in the past month

[0058] Chronic intestinal disorder

[0059] Particular dietary regimen (i.e. vegetarian)

[0060] Diagnosis of gastrointestinal cancer

[0061] Presence of flatulence

[0062] Approval was obtained from the institutional ethical committee.Written informed consent was obtained from all subjects. The study wascarried out at a local nursing home, Le Mont-Pelerin, Switzerland.

[0063] Nutritional Status

[0064] At entrance into study nutritional status was evaluated by theMini Nutritional Assessemnt (MNA) test which includes the followingitems: anthropometric measurements (calf and arm circumference, height,weight and weight loss), general assessments (lifestyle, medications,mobility), dietary questionnaires (number of meals, fluid and foodintakes, autonomy of feeding) and subjective assessments (selfperception for health and nutrition.) [Guigoz, Y., et al. Nutr Rev1996;54:S59-565]. MNA classified the elderly nutritional status using a30 point scale: MNA≧24=wellnourished, MNA 17-23.5=at risk ofmalnutrition and MNA<17=malnourished.

[0065] Prebiotic Supplementation

[0066] Eight gram of short chain fructooligosaccharides (FOS) per daywas administered as follows: twice a day 4 g FOS powder (Actilight 950P,Bëghin-Meiji industries, Neuilly-sur-Seine, France) were incorporated ina dish at the time of the meal by the nurses. For estimation ofcompliance, the daily consumption of the supplement was recorded by thenurses on a daily record sheet.

[0067] Microbial Investigations

[0068] The endogenous populations of Lactobacilli, Bacteroïdes,Enterobacteriaceae, Enterococci, Bifidobacteria and Clostridiumpeifringens were counted.

[0069] Stool samples were collected at day 0, 21, 42 and 63 from eachsubject. The stool samples were immediately (within 30 minutes) placedin an anaerobic jar and kept at 4° C. until analysis (a maximum of 6hours). Hundred fold serial dilutions were performed in pre-reducedRinger solution containing 0.5% of cystein, from −2 to −8. Petri dishesof various media were inoculated and incubated for 48 hr at 37° C. inanaerobic atmosphere using Anaerocult A (Merck, Darmstadt, Germany),except for Enterococci and Enterobacteriacea incubated for 24 hr at 37°C. in aerobic atmosphere. Bacteria were detected on selective orhalf-selective media as follows: Enterobacteriacea on Drigalski medium(Sanofi Diagnostics Pasteur, France), Bifidobacteria on Eugon Tomatomedium (Wadsworth Anaerobic Bacteriology Manual, V. Suter, D. Citron andS. Finegold Third edition), Lactobacilli in MRS (Difco, MI. USA) withantibiotics (Phosphomycine (79.5 mg/l)+Sulfamethoxazole (0.93mg/l)+Trimethoprime (5 mg/l)), Clostridium perfringens on NN agar(Lowbury and Lilly, 1995), Bacteroïdes on Schaedler Neo-Vanco medium(BioMérieux, Marcy-I'Etoile, France), and Enterococci on Azide agar(Difco).

[0070] After incubation, the colonies were counted and furtheridentified if necessary. Lactobacilli and Bifidobacteria strains wereidentified by microscopy, and biochemically using the API gallery system(BioMérieux), API 50 CHL gallery for lactobacilli, and API ID 32Agallery for Bifidobacteria respectively. Bacterial counts are expressedas log₁₀ colony-forming units (CFUs) per gram of fresh faecal sample,with a detection limit at 3.30 cfu/g.

[0071] Faecal PH

[0072] Faecal pH was measured just after emission by the nurse at threedifferent points in crude faeces with a microelectrode (Orion). The meanpH was calculated for each time point.

[0073] Blood Sampling

[0074] Fasting blood samples were drawn before the start of the study (4ml) and at day 0, 21, 42 and 63 (23 ml) from each subject. Blood wastaken in the morning (before 10 a.m.) after an overnight fast. Samplescollected in vacutainer tubes. 13 ml were collected in heparinized tube,and 10 ml were allowed to clot at room temperature for 30-60 minutes.Serum samples were kept frozen at −70° C. until analysis. Heparinizedblood was used for the functional immune parameters (phagocytosis, seebelow) and hematology.

[0075] Immunological Analysis

[0076] Populations of peripheral blood mononuclear cells and theirphagocytic activity were analyzed in fresh samples of heparinized bloodusing Simulset and Pagotest (Becton Dickinson, Basel, Switzerland).

[0077] Analysis of interleukin-6 mRNA expression by PCR: Analysis ofmRNA expression in the peripheral blood mononuclear cells was evaluatedby PCR according to the method described by Delneste, Y., et al. (NutrRev 1998; 56:S93-S98). Primer sequences were:5′-CTGCAGGAACTGGATCAGGACTTTTGTACT-3′ and5′-GCCTTCGGTCCAGTTGCCTTCTCCCTGGGG-3′ for interleukin-6 (IL-6) and5′CGTTTCCCGCTCGGCCGTGGTGGTGAAGC-3′ and5′-GGCGACGAGGCCCAGAGCAAGAGAGGCATC-3′ for β-actin.

[0078] Biochemical Measurements

[0079] Serum albumin, transthyretin (prealbumin), C-reactive protein,and a, acid glycoprotein concentrations were analyzed byimmuno-nephelometry with a Behring Nephelometer (methods and reagentsfrom Behring, Marburg, Germany). Serum folate and vitamin B₁₂(cobalamin) concentrations were analyzed by radioimmunoassay (DualCount, Diagnostic Product Corporation, Los Angeles, Calif., USA).

[0080] Statistical Analysis

[0081] One sample t-test or Wilcoxon signed-rank test were computed onthe mean differences to evaluate the statistical significance at 0.05%level for the main hypothesis: Is there an effect of prebiotics on thedifferent parameters measured? Further one sample t-test were computedon-the mean differences to evaluate the statistical significance at0.025% level (correction for the loss of degree of freedom) for the twoquestion related to the design of the study, which included pre and posttest periods: Is there an effect of stopping fermented milk (=wash-outperiod) on the different parameters measured? Is there an effect ofstopping prebiotics (=follow-up period) on the different parametersmeasured? All statistical analysis were done using the NCSS 6.0.22statistical software.

[0082] Results

[0083] Subjects

[0084] The 19 subjects, 4 men and 15 women, had a mean age of 85±6.0years (77-97 year old). Women were significantly lighter than men.Subjects' characteristics at start of study are given in table 1. TABLE1 Subjects characteristics 95% confidence Range mean interval min MaxMen n = 4 Age [year] 84 5.7 77 91 Body weight [kg] 72.1 13.8 60.4 92.4BMI [kg/mE2] 27.5 5.5 23.4 35.7 MNA score [pts] 27.0 2.9 24.0 30.0Albumin [g/L] 36.3 1.7 31.1 40.9 Transthyretin [g/L] 0.23 0.04 0.17 0.28α1-acid glycoprotein [g/L] 0.98 0.16 0.82 1.20 C-reactive protein [mg/L]15 12 3 28 Cholesterol [mmol/l] 4.23 1.26 2.92 6.00 Triglycerides[mmol/l] 1.39 0.59 0.65 1.99 Phospholipids [mmol/l] 2.10 0.51 1.52 2.78Women n = 15 Age [year] 85 3.1 77 97 Body weight [kg] 57.5 5.8 39.7 80.0BMI [kg/mE2] 25.7 1.7 17.5 30.7 MNA score [pts] 23.9 1.4 17.0 28.0Albumin [g/L] 36.8 1.7 31.1 42.7 Transthyretin [g/L] 0.22 0.02 0.15 0.23α1-acid glycoprotein [g/L] 0.85 0.10 0.56 1.19 C-reactive protein [mg/L]5 2 1 15 Cholesterol [mmol/l] 5.72 0.56 3.81 7.69 Triglycerides [mmol/l]1.51 0.23 1.11 2.55 Phospholipids [mmol/l] 2.55 0.18 2.03 3.21

[0085] Only one subject was malnourished, the 7 women at risk ofmalnutrition were in the upper-score range 21.5-23, and the mean MNAscore was in the wellnourished range: 24.6±3.0 points.

[0086] Serum albumin (normal range 35-55 g/L) and transthyretin(prealbumin; normal range 0.16-0.40) were in the lower normal range,while the acute phase proteins, α1-acid glycoprotein (normal range0.5-1.3) and C-reactive protein (normal values <10 mg/L and abnormalvalues for elderly >20 mg/L) indicated no presence of inflammatoryprocess, except for 2 men (elevated C-reactive protein levels) and 2women (low albumin levels with normal transthyretin and borderlineC-reactive protein). These results suggest a group of elderly who werestill wellnourished but rather frail.

[0087] Bacteriological Analyses and Faecal PH

[0088] Bacterial counts of bifidobacteria were increased by a mean of2.8±0.57 log₁₀CFU during the 3 weeks of FOS supplementation (p<0.001),but Bacteroïdes counts were also increased p<0.032) (see tables 2a & 2band FIG. 1). TABLE 2a Effect of FOS administration on faecal flora andpH Pretest Before During After administration of fructooligosaccharides(8 g/day) Faecal mean 95% CI mean 95% CI mean 95% CI mean 95% CIvariable log₁₀CFU/g faeces Enterobacteriaceae 7.7 0.6 7.2 0.8 7.1 0.98.0 0.7 Enterococci 6.1 0.8 5.7 0.9 5.4 0.8 6.2 0.9 Bifidobacteria 6.01.1 5.6 1.2 8.4 1.0 7.3 1.2 Lactobacilli 5.1 0.6 5.1 0.6 5.7 1.0 5.1 0.7Bacteroides 8.8 0.3 8.6 0.3 9.3 0.3 9.8 0.3 Clostridium 3.5 0.2 3.7 0.53.7 0.5 3.5 0.4 perfringens pH pH 7.1 0.2 6.9 0.2 7.0 0.2 7.0 0.2

[0089] Bacterial counts for Enterobacteriaceae, Enterococci, andlactobacilli were not significantly affected by suppression of fermentedmilk products and/or the ingestion of fructooligosaccharides (FOS).While for bifidobacteria the effect seems to be a specific response tothe ingestion of FOS, it resulted mainly in increased counts ofbifidobacteria for subjects showing a log₁₀ CFU lower than 7 beforestarting FOS. Suppression of FOS supplementation significantly decreasedcounts of bifidobacteria by 1.1±0.39 log₁₀ CFU, but not to start level(tables 2a & 2b and FIG. 1). In FIG. 2 changes in bifidobacteriaobtained in this study are compared to previous studies indicating thatelderly are at least as sensitive to the effect of FOS as youngeradults. Neither the suppression of fermented milk products nor theingestion of FOS changed faecal pH (table 2a).

[0090] Non-Specific Immunity

[0091] Ingestion of FOS resulted in a significant increased percentageof peripheral T lymphocytes as well as the lymphocyte subsets, CD4+,CD8+ T cells (tables 3a & 3b). Total number of white blood cells,activated T lymphocytes and natural killer (NK) cells were not affectedby the ingestion of FOS (tables 3a & 3b). TABLE 3a Peripheralimmunological parameters: lymphocyte subpopulations Pretest BeforeDuring After Administration of fructooligosaccharides (8 g/day) VariableUnits Mean 95% CI Mean 95% CI Mean 95% CI Mean 95% CI White blood cells# 10⁻³ nd 4.9 0.6 5.3 0.7 5.8 0.7 T lymphocytes % 65.7 4.8 64.0 4.3 68.75.4 66.9 4.1 B lymphocytes %  7.7 1.5 8.3 1.7 8.5 1.4 8.1 1.5 CD 4⁺cells% 40.8 4.4 41.7 4.1 47.3 4.5 45.1 4.0 CD 8⁺cells % 35.3 4.4 33.2 4.538.3 4.7 36.8 4.7 Activated T % 15.0 3.8 15.7 4.1 15.6 4.7 18.6 5.3lymphocytes NK cells % 22.0 3.9 21.9 3.4 24.8 3.4 21.3 3.3

[0092] TABLE 3b-c-d Changes in peripheral immunological parameters Datagiven are the mean difference ± standard error of the mean (sem) aftereach period of 3 weeks No fermented milk Products (Wash-out period) n =13-19 units Mean Δ² ±sem p value⁵ White blood cells # 10⁻³ — — — Tlymphocytes % −1.7 0.97 0.157 B lymphocytes % 0.4 0.34 0.298 CD 4⁺ cells% 0.8 0.91 0.397 CD 8⁺ cells % −2.1 0.82 0.019 Activated T lymphocytes %0.7 0.72 0.353 NK cells % 0.05 1.15 0.964 FOS¹ 8 g/day (Prebiotic intakeperiod) n = 13-19 units Mean Δ³ ±sem p value⁵ White blood cells # 10⁻³0.347 0.286 0.243 T lymphocytes % 4.6 1.49 0.006 B lymphocytes % 0.30.44 0.562 CD 4⁺ cells % 5.7 1.34 <0.001 CD 8⁺ cells % 5.0 0.99 <0.001Activated T lymphocytes % −0.1 1.00 0.918 NK cells % 2.9 1.48 0.066 NoFOS (Follow-up period) n = 13-19 units Mean Δ⁴ ±sem p value⁵ White bloodcells #10⁻³ 0.495 0.362 0.326 T lymphocytes % −1.8 1.42 0.225 Blymphocytes % −0.4 0.46 0.434 CD 4⁺ cells % −2.3 1.38 0.111 CD 8⁺ cells% −1.4 1.14 0.227 Activated T lymphocytes % 3.0 0.93 0.005 NK cells %−3.6 1.40 0.020

[0093] Phagocytic activity of granulocytes and monocytes weresignificantly decreased by the ingestion of FOS: Phagocytic activityexpressed as median fluorescent intensity changed for granulocytes from130±10 to 52±2 (p<0.001) and for monocytes from 75±5 to 26±2 (p<0.001).

[0094] This possible decrease in inflammatory process is also suggestedby the significant decrease in Interleukin-6 mRNA levels in peripheralblood mononuclear cells after ingestion of FOS (FIG. 3).

[0095] Vitamin B₁₂ and Folate Status

[0096] Neither vitamin B₁₂ nor folate serum levels were influenced bythe supplementation in FOS: Serum vitamin B₁₂ were at 271±143 ng/Lbefore supplementation and at 289±160 ng/L during supplementation. Threesubjects, however, were deficient in vitamin B₁₂. Two subjects returnedto normal status during the study while the other remained deficientthroughout study. Serum folate levels were at 5.9±2.0 ug/L beforesupplementation and at 5.7±1.9 ug/L during supplementation.

[0097] Our results strongly support the bifidogenic effects offructooligosaccharides in elderly subjects with a 2 log increase inbifidobacteria counts since our frail elderly subjects showed low countsat the beginning of the study. A diminution in inflammatory process issuggested by the decreased expression of IL-6 mRNA in peripheral bloodmonocytes. Indeed, the present study confirms the positive effect of FOSsupplementation on bifidobacteria observed in adults and elderly (FIG.2), indicating that elderly respond to prebiotic (FOS) intake by anincrease in bifidobacteria like younger adults or even better if thebifidobacteria counts are low. Further 8 g of FOS or less seems to besufficient to achieve a maximal effect on bifidobacteria counts. Whilefrom different studies a dose response seems to be present, singlestudies indicate that above a threshold of 4-5 g/day a maximal responseis obtained and the increase in bifidobacteria seems to be moredependent of the initial number (FIGS. 1 and 2). Often FOS added to thediet increased the levels of bifidobacteria at the expense ofpotentially harmful bacteria, clostridia and Bacteroïdes mainly. But weobserved a significant increase in Bacteroïdes throughout the study(tables 2a & 2b).

[0098] We observed an important decrease in phagocytic activity. Thisdecrease in phagocytic activity could be a reflection of decreasedactivation of macrophages linked to a possible reduction in pathogenicbacteria, and thus suggesting a diminution in inflammation due to lowerendotoxin load. Surprisingly, however, this possible decrease ininflammatory process is suggested by the decrease in Interleukin-6 mRNAlevels in peripheral blood mononuclear cells (FIG. 3).

EXAMPLE 2 Food Supplement

[0099] A food supplement was prepared by mixing or blendingfructooligosaccharide with inulin in the proportions by weight of about70% fructooligosaccharide to about 30% inulin. The resulting prebioticmixture may be added or blended with any suitable carrier, for example afermented milk, a yogurt, a fresh cheese, a renneted milk, aconfectionery bar, breakfast cereal flakes or bars, a drink, milkpowder, soy-based product, non-milk fermented product or a nutritionalsupplement for clinical nutrition.

EXAMPLE 3 Dry Pet Food

[0100] A feed mixture is made up of about 58% by weight of corn, about5.5% by weight of corn gluten, about 22% by weight of chicken meal, 2,5%dried chicory, 1% carnitine, salts, vitamins and minerals making up theremainder.

[0101] The fed mixture is fed into a preconditioner and moistened. Themoistened feed is then fed into an extruder-cooker and gelatinised. Thegelatinised matrix leaving the extruder is forced through a die andextruded. The extrudate is cut into pieces suitable for feeding to dogs,dried at about 110° C. for about 20 minutes, and cooled to form pellets.

[0102] This dry dog food is particularly intended for decreasinginflammatory process and/or abnormal activation of non specific immuneparameters, such as phagocytes.

EXAMPLE 4 Wet Canned Pet Food

[0103] A mixture is prepared from 56% of poultry carcass, pig lungs andpig liver (ground), 13% of fish, 16% of wheat flour, 2% of plasma, 10.8%of water, 2.2% of dyes, 1% of semi refined kappa carrageenan, inorganicsalts and 9% oil rich in monounsaturated fatty acids (olive oil) and 3%chicory. This mixture is emulsified at 12° C. and extruded in the formof a pudding which is then cooked at a temperature of 90° C. It iscooled to 30° C. and cut in chunks.

[0104] 30% of these chunks (having a water content of 58%) isincorporated in a base prepared from 23% of poultry carcass, 1% of guargum, 1% of dye and aroma and 75% of water. Tinplate cans are then filledand sterilized at 127° C. for 60 min.

1. A composition comprising at least one prebiotic in an amountsufficient so as to decrease inflammatory process and/or abnormalactivation of non specific immune parameters in a mammal consuming thecomposition.
 2. A composition according to claim 1, wherein thecomposition decreases abnormal activation of phagocytes in the mammal.3. A composition according to claim 1, wherein the prebiotic comprisesan oligosaccharide produced from a component selected from the groupconsisting of glucose, galactose, xylose, maltose, sucrose, lactose,starch, xylan, hemicellulose, inulin, gum and a mixture thereof.
 4. Acomposition according to claim 1, wherein the prebiotic comprises afructooligosaccharide.
 5. A composition according to claim 1, whereinthe prebiotic comprises a mixture of fructooligosaccharide and inulin.6. A composition according to claim 1, wherein the prebiotic comprises,by weight, about 60% to about 80% fructooligosaccharide and about 20% toabout 40% inulin.
 7. A composition according to claim 1 which comprisesa probiotic.
 8. A composition according to claim 7 wherein the probioticis selected from the group consisting of bifidobacterium bifidum andstreptococcus thermophilus.
 9. A composition according to claim 1,designed to treat a patient selected from the group consisting ofelderly humans, elderly pets, and critically ill patients having chronicinflammation.
 10. A method of manufacturing a or pet food compositionfor decreasing inflammatory process in a mammal comprising the steps ofusing at least one prebiotic.
 11. The method of claim 10, wherein asufficient amount of prebiotic is used in the manufature of thecomposition to provide a product for decreasing abnormal activation ofnon-specific immune parameters in a mammal.
 12. The method according toclaim 10, wherein the prebiotic comprises an oligosachharide producedfrom a component selected from the group consisting of glucose,galactose, xylose, maltose, sucrose, lactose, starch, xylan,hemicellulose, inulin, gum and a mixture thereof.
 13. The methodaccording to claim 10, wherein the prebiotic comprises afructooligosaccharide.
 14. The method according to claim 10, wherein theprebiotic comprises, by weight, about 60% to about 80%fructooligosaccharide and about 20% to about 40% inulin.
 15. The methodaccording to claim 10, wherein the prebiotic is provided in a mixturewith a probiotic.
 16. The method according to claim 10, wherein theprobiotic is selected from the group consisting of Bifidobacteriumbifidum or Streptococcus thermophilus.
 17. A method of decreasinginflammatory process in a mammal comprises comprising administering aneffective amount of a prebiotic to the mammal.
 18. A method ofdecreasing the abnormal activation of non-specific immune parameters ina mammal, comprising the steps of administering an effective amount of aprebiotic.
 19. A method according to claim 17, wherein the prebioticcomprises an oligosaccharide produced from a component selected from thegroup consisting of glucose, galactose, xylose, maltose, sucrose,lactose, starch, xylan, hemicellulose, inulin, gum and a mixturethereof.
 20. A method according to claim 17, wherein the prebioticcomprises a fructooligosaccharide.
 21. A method according to claim 17,wherein the prebiotic comprises a mixture of fructooligosaccharide andinulin.
 22. A method according to claim 17 wherein the prebioticcomprises, by weight, about 60% to about 80% fructooligosaccharide andabout 20% to about 40% inulin.
 23. A method according to claim 17, whichcomprises administering of an effective amount of a probiotic.
 24. Amethod according to claim 17 wherein the prebiotic is in a compositionhaving other components.
 25. A method according to claim 18 wherein theprebiotic is in a composition having other components.
 26. A methodaccording to claim 18, wherein the prebiotic comprises afructooligosaccharide.
 27. A method according to claim 18, wherein theprebiotic comprises a mixture of fructooligosaccharide and inulin.
 28. Amethod according to claim 18 wherein the prebiotic comprises, by weight,about 60% to about 80% fructooligosaccharide and about 20% to about 40%inulin.
 29. A method according to claim 18, which comprisesadministering an effective amount of a probiotic.
 30. A method accordingto claim 18 wherein the prebiotic is in a composition having othercomponents.